Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragmentsfor tumor targeting

Efficient T-cell activation requires two signals. The first signal, which c onfers specificity, is provided hy interaction of the T-cell receptor with peptides presented by MHC molecules, One of the second costimulatory signal s is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor c ells can result in the activation of tumor specific T lymphocytes and induc e protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expre ssion to cancer cells, As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface o f tumor cells. We constructed chimeric proteins that contain in a single po lypeptide chain a portion of human B7-2 (CD86) genetically fused to single- chain (sc) Fv antibody domains specific for the tumor-associated antigens e pidermal growth factor receptor and the closely related ErbB2 receptor tyro sine kinase, A small recombinant fragment of human CD86 was characterized t hat corresponds to amino acid residues 1-111 (CD86(111)) of the mature prot ein. CD86(111) produced in the yeast Pichia pastoris and CD86(111) expresse d in bacteria was functionally active and displayed specific binding to B7 counter receptors, Bacterially expressed CD86(111)-scFv fusion proteins spe cifically localized to the respective target antigens on the surface of tum or cells and markedly enhanced the proliferation of primary T cells when bo und to immobilized tumor antigen.